ORIGINaL PaPERS - Advances in Clinical and Experimental Medicine

original papers
Adv Clin Exp Med 2013, 22, 3, 355–359
ISSN 1899–5276
© Copyright by Wroclaw Medical University
Elisabeth Brunnerc, d, Karla Lehlea, f, Stephan W. Hirta, e, Christof Schmide, f,
Marietta von Suesskind-Schwendia, c, d
Everolimus’s Influence on Persistent Acute Rejection
After Experimental Lung Transplantation*
Wpływ ewerolimusu na przewlekłe ostre odrzucenie doświadczalnego
przeszczepu płuc
Department of Cardiothoracic Surgery, University Medical Center Regensburg, University of Regensburg,
Germany
A – research concept and design; B – collection and/or assembly of data; C – data analysis and interpretation;
D – writing the article; E – critical revision of the article; F – final approval of article; G – other
Abstract
Background. In lung transplantation, acute rejection episodes increase the risk of chronic rejection. Therefore
treatment of acute rejection needs to be optimized for better long-term outcome of lung-transplantation and
patient survival.
Objectives. The aim was to verify whether an inhibitor of the mammalian target of rapamycin (Everolimus) contained the extent of persistent acute rejection after left lung allo-transplantation in rats.
Material and Methods. Rats (F344-to-WKY) with a high grade of acute rejection were treated with methylprednisolone (10 mg/kg, postoperative days 14–16) alone or in combination with everolimus (2.5 mg/kg, postoperative
days 14–30). The rats were killed on postoperative day 20 and 30. Infiltration of inflammatory cells (ED1, CD11a,
CD18) and activation of endothelial cells (ICAM-1) were measured by immunohistochemistry
Results. Everolimus treatment significantly reduced the number of ICAM-1 positive small vessels (66%; p < 0.05)
and suppressed the infiltration of leucocytes (CD11a (64%), CD18 (42%); p < 0.05) and macrophages (ED1; 22%)
in the allografts on POD 30. Despite this clear anti-inflammatory effects, lung allografts still showed severe acute
vascular rejection in combination with high grade small airway inflammation.
Conclusions. The shown anti-inflammatory effects of Everolimus could not delay the progression of acute rejection in rat lung allografts (Adv Clin Exp Med 2013, 22, 3, 355–359).
Key words: lung transplantation, acute lung injury, Everolimus, rats, rejection, inflammation.
Streszczenie
Wprowadzenie. Ostre odrzucenie przeszczepionego płuca zwiększa ryzyko przewlekłego odrzucenia. Dlatego
leczenie ostrego odrzucenia musi być zoptymalizowane, aby osiągnąć lepszy długoterminowy wynik przeszczepienia płuc i przeżycia pacjenta.
Cel pracy. Sprawdzenie, czy inhibitor ssaczego celu rapamycyny (ewerolimus) chroni przed trwałym ostrym
odrzuceniem po allogenicznym przeszczepieniu lewego płuca u szczurów.
Materiał i metody. Szczury (F344-WKY) o dużym stopniu ostrego odrzucania leczono metyloprednizolonem
(10 mg/kg, dni po operacji 14–16) samym lub w połączeniu z ewerolimusem (2,5 mg/kg, dni po operacji 14–30).
Szczury zabito w 20. i 30. dniu po operacji. Naciek komórek zapalnych (ED1, CD11a, CD18) i aktywację komórek
śródbłonka (ICAM-1) mierzono metodą immunohistochemiczną.
Wyniki. Leczenie ewerolimusem znacząco zmniejszyło liczbę ICAM-1 dodatnich drobnych naczyń (66%, p < 0,05)
i ograniczyło naciek leukocytów (CD11a (64%), CD18 (42%), p < 0,05) i makrofagów (ED1, 22%) w przeszczepach
allogenicznych na POD 30. Mimo że allogeniczne przeszczepy płuc były pod wpływem wyraźnego działania przeciwzapalnego, nadal wykazywały ciężkie ostre odrzucenie naczyniowe w połączeniu z zaawansowanym zapaleniem
drobnych dróg oddechowych.
* SH supported by a grant from the Deutsche Forschungsgemeinschaft (No. HI-1333). MvS was founded by a grant
from University of Regensburg (No. I101-03).
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E. Brunner et al.
Wnioski. Wykazane przeciwzapalne działanie ewerolimusu nie opóźnia rozwoju ostrego odrzucenia przeszczepów
płuca szczura (Adv Clin Exp Med 2013, 22, 3, 355–359).
Słowa kluczowe: przeszczepienie płuc, ostra niewydolność oddechowa, ewerolimus, szczury, odrzucenie, zapalenie.
Lung transplantation (LTx) is the therapy of
choice for a variety of end stage pulmonary diseases. Despite advances in immunosuppressive
therapy, acute allograft rejection and lymphocytic
bronchiolitis still cause chronic allograft rejection,
morbidity and mortality [1]. While the standard
short-term steroid therapy can contain acute allograft rejection, these patients often develop recurrent acute rejection or persistent rejection. The
underlying mechanisms are unknown [2]. Meanwhile, it is clear that immunosuppressive drugs can
spare or even induce certain immune cells. Such
drug specific effects may be responsible for acute
persistent-recurrent rejection [1]. The use of certain immunosuppressants, like mTOR inhibitors
(mammalian target of rapamycin, e.g. Everolimus)
could optimize transplantation outcome.
Today, Everolimus is successfully used to prevent acute and chronic rejection after solid organ
transplantation [3]. The authors hypothesized
Everolimus [4] should inhibit the progression of
persistent acute rejection after left lung allo-transplantation in rats by reducing graft infiltration inflammatory cells (ED1+, CD11a+, CD18+) and by
inhibiting expression of the endothelial activation
marker ICAM on graft vasculature.
Material and Methods
In the moderate allogeneic F344-to-WKY
(n = 20) rat strain combination, both acute rejection in the short term after LTx (up to postoperative day (POD) 30) and chronic rejection
in the long term follow up can be analyzed [5].
Orthotopic left LTx was performed as described
earlier [5]. The right lobes remained in the recipient and served as internal controls. The rats were
purchased from Charles River (Sulzfeld, Germany;
280±30g). All animals received humane care in
compliance with the European Union Guide for
the Care and Use of Laboratory Animals (publication No. 86/609/EWG) and permission from
the local authorities was obtained. In order to test
treatment options for acute rejection, rats in the
control group (n = 10) received intraperitoneal
methylprednisolone (MP, Urbason®, Hoechst AG,
Frankfurt, Germany; 10 mg/kg body weight) injections on POD 14–16. The rats in the Everolimusgroup (n = 10) additionally received Everolimus
(Novartis Pharma, Basel, Switzerland; 2.5 mg/kg
body weight, intragastral on POD 14–30). Cohorts
of 5 animals each were killed on POD 20 and 30.
The right lungs were used as internal controls. The
native lungs of non-transplanted rats were used
as reference. In addition to conventional histology (H/E staining), immune histological staining
of inflammatory and cell adhesion markers (ED1,
CD11a, CD18, ICAM-1) was analyzed [6]. All antibodies were purchased from BD Pharmingen (Heidelberg, Germany). Acute rejection was graded according to the ISHLT working formulation [7].
The number of ED1+, CD11a+ and CD18+
cells and the proportion of ICAM-1+ vessels was
measured in five high power fields (HPF). ICAM-1
was graded: 1: < 25%, 2: 25–50%, 3: 50–75%,
4: > 75% positive vessels per HPF. The difference
between the left and right lung of each animal was
used to quantify the drug effect on acute rejection.
Statistical analysis (SPSS 16.0.2, SPSS inc, Chicago,
IL) included analysis of variance (ANOVA) followed by a Dunnett T3 post-hoc-test with p-values
considered significant below < 0.05. Cell numbers
and graded ICAM positive vessels are presented as
mean +/– STD per HPF.
Results
All allografts from both the control group and
the Everolimus group showed moderate to severe acute cellular rejection and high grade lymphocytic bronchiolitis (Table 1) on POD 20 and
POD 30. The right lungs of the transplanted rats
showed normal, non-inflammatory cell numbers.
(ED1, 17 ± 1; CD11a, 24 ± 4; CD18, 27 ± 2; proportion of ICAM-1+ vessels, 2.3 ± 0.3). Similar results were documented in the native lungs of nontransplanted rats (data not shown). Allogeneic
LTx caused an increased infiltration of inflammatory cells and induced activated endothelial cells
in small vessels. The number of inflammatory cells
and the proportion of activated vessels in the allografts significantly increased within 20 days after
LTx in both groups, without showing a significant
difference due to Everolimus (increase in the allograft (vs. right lung): control-group: ED1: 8 ± 2;
CD11a, 16 ± 2; CD18, 27 ± 5; ICAM-1: 1.5 ± 0.3;
Everolimus-group: ED1: 7 ± 3; CD11a, 19 ± 2;
CD18, 28 ± 2; ICAM-1, 1.0 ± 0.7). On POD 30, the
inflammatory response remained high in the control group. Continued treatment with Everolimus
resulted in a significant decrease in the number of
infiltrated lymphocytes (CD11a+ and CD18+ cells,
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Rat Lung Transplantation and Everolimus
Fig. 1. Evaluation of lymphocytic cells (CD11a, CD18), macrophages (ED1) and activated vessels (ICAM-1) during
severe acute rejection (POD 30). White column: MP control group, black column: Everolimus group. There was a significant reduction of lymphocytic cells and activated vessels (*p < 0.05, error bar: SEM). Cell number normalized to right
(non-transplanted) lung. (ICAM-1 was graded: 1: < 25%, 2: 25–50%, 3: 50–75%, 4 > 75% positive vessels per HPF)
Ryc. 1. Ocena komórek limfocytarnych (CD11a, CD18), makrofagów (ED1) i aktywowanych naczyń (ICAM-1) podczas ciężkiego ostrego odrzucenia (POD 30). Biała kolumna: grupa kontrolna, czarna kolumna: grupa leczona ewerolimusem. Stwierdzono istotne zmniejszenie liczby komórek limfocytarnych i aktywowanych naczyń (*p < 0,05; błąd:
SEM). Liczba komórek była znormalizowana do prawego płuca (nieprzeszczepionego). (ICAM-1 oceniano: 1: < 25%,
2: 25–50%, 3: 50–75%, 4 > 75% pozytywnych naczyń na HPF)
Table 1. Evaluation of acute vascular rejection and lymphocytic bronchiolitis (B) in control and Everolimus groups. Samples
were graded according to the International Society for Heart and Lung Transplantation (ISHLT) [7]. (A0: no acute vascular
rejection to A4: severe acute vascular rejection; B0: no airway inflammation to B2R: high-grade airway inflammation)
Tabela 1. Ocena ostrego naczyniowego odrzucenia i limfocytarnego zapalenia oskrzelików (B) w grupie kontrolnej i leczonej
ewerolimusem. Próbki były klasyfikowane według Międzynarodowego Towarzystwa Transplantacji Serca i Płuc (ISHLT) [7].
(A0: brak ostrego odrzucenia do A4: naczyniowe ciężkie ostre naczyniowe odrzucenie; B0: brak zapalenia dróg oddechowych
do B2R: zapalenie dróg oddechowych ciężkiego stopnia)
Control
(Grupa kontrolna)
Everolimus
(Grupa leczona ewerolimusem)
ISHLT-grade (mean)
ISHLT-grade (mean)
POD 20
A4/B2R (n = 5)
A4/B1R-B2R (n = 5)
POD 30
A4/B2R (n = 5)
A3-4/B1R-B2R (n = 5)
p ≤ 0.05) on POD30, as shown in Figure 1. The proportion of activated vessels was significantly lower
in the allografts of the Everolimus group (p ≤ 0.05),
while the number of alveolar macrophages (ED1)
remained unchanged. However, the decrease in the
number of infiltrated lymphocytes does not result
in a decrease of the ISHLT-scoring of acute cellular
rejection and lymphocytic bronchiolitis (Table 1).
Discussion
Persistent and recurrent acute rejection episodes are major risk factors in lung transplant patients leading to chronic rejection and graft failure. The exact trigger remains unknown. Present
data shows that the anti-inflammatory properties
of Everolimus cause a significant containment of
inflammatory infiltration in allografts after experimental LTx.
The relevance of acute rejection as a risk factor for the development of chronic rejection was
already shown in the same rat left lung allo-transplantation model (F344-to-WKY) by Hirt et al. [8].
Both early (POD 9–11) and late (POD 14–16) application of a steroid bolus attenuated the extent
of acute rejection on POD 20. However, only early
therapy suppressed chronic rejection in the longterm follow-up. Late treatment of acute rejection
could not prevent the development of extensive
vascular fibrosis and bronchiolitis obliterans [8].
However, in experimental rats, late application of
MP on POD 14–16 did not affect acute rejection.
The reasons for this discrepancy might be breed-
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E. Brunner et al.
ing differences resulting in various sublines of the
same rat strain [5]. Hirt et al. used animals from
Harlan-Winkelmann and the experimental rats
were purchased from Charles River. The source
and husbandry history of the animals is important
for analysis of immuno-mediated processes [5].
In the present study the authors have used their
more stringent model to test the hypothesis that
an inhibitor of the mammalian target of rapamycin (Everolimus) can rescue steroid resistant
acute rejection. In present model, POD 14 reflects
the zenith of acute rejection and simulates a persistent acute rejection [5]. In order to simulate
the clinical situation of an occurring acute rejection, the authors started Everolimus therapy on
POD14. Moreover, Everolimus should be used
with care early after transplantation, since it can
cause severe side effects upon surgery (e.g. wound
healing complications or weight loss) [10]. In the
present model, Everolimus significantly reduced
the infiltration of lymphocytes (CD11a, CD18)
and reduced the expression of its corresponding
endothelial receptor (ICAM-1) in small vessels of
lung allografts. The underlying mechanisms might
be the suppression of inflammatory mediators (IL6, MCP-1) and T-cell activating cytokines (IL-2,
IFN-gamma) [9], as well as the inhibition of the
growth factor-stimulated proliferation of lymphocytes [4]. Despite these encouraging findings, this
Everolimus treatment did not hinder progression
to severe cellular rejection and advanced lymphocytic bronchiolitis in the present model. The histological evaluation of the allografts described an
ISHLT-A4/B2R in both groups on both POD 20
and 30. Based on the high grade of acute rejection,
the authors suppose that treatment of persistent
acute rejection with a bolus of MP and Everolimus
could not prevent the development of chronic rejection later on. Similar results were already shown
in an earlier study using a monotherapy of Everolimus [10], where the authors could show that only
early application of Everolimus reduced the grade
of acute rejection and the development of vasculopathy and bronchiolitis obliterans [10].
In conclusion, the anti-inflammatory effects
of Everolimus are – even in combination with steroids – not sufficient to delay the progression of
acute rejection in lung allografts. Summing up two
of authors’ animal studies, they hypothesize that
patients would benefit from Everolimus only if
therapy is started before the occurrence of rejection episodes.
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Rat Lung Transplantation and Everolimus
Address for correspondence:
Marietta von Suesskind-Schwendi
Tel.: +49-(0)941-944 9901
E-mail: [email protected]
Conflict of interest: None declared
Received: 20.01.2013
Revised: 15.03.2013
Accepted: 13.06.2013
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